Aryl hydrocarbon receptor agonist indigo protects against obesity-related insulin resistance through modulation of intestinal and metabolic tissue immunity

International Journal of Obesity https://doi.org/10.1038/s41366-019-0340-1 ARTICLE Aryl hydrocarbon receptor agonist indigo protects against obesity-related insulin resistance through modulation of intestinal and metabolic tissue immunity Yi-Hsuan Lin1,2,3,4 Helen Luck4,5 Saad Khan4,5 Pierre H. H. Schneeberger6,7 Sue Tsai4 Xavier Clemente-Casares4 Helena Lei4 Yann-Lii Leu1,8 Yi Tao Chan4,5 Hsing-Yu Chen1,2,3 Sien-Hung Yang1,2,9 Bryan Coburn6,7 Shawn Winer4,7,10 Daniel A. Winer4,5,7,11,12,13 ● ● ● ● ● ● ● ● ● ● ● ● ● 1234567890();,: 1234567890();,: Received: 9 August 2018 / Revised: 24 December 2018 / Accepted: 19 February 2019 © The Author(s) 2019. This article is published with open access Abstract Background/objectives Low-grade chronic inflammation in visceral adipose tissue and the intestines are important drivers of obesity associated insulin resistance. Bioactive compounds derived from plants are an important source of potential novel therapies for the treatment of chronic diseases. In search for new immune based treatments of obesity associated insulin resistance, we screened for tissue relevant anti-inflammatory properties in 20 plant-based extracts. Methods We screened 20 plant-based extracts to assess for preferential production of IL-10 compared to TNFα, specifically targetting metabolic tissues, including the visceral adipose tissue. We assessed the therapeutic potential of the strongest antiinflammatory compound, indigo, in the C57BL/6J diet-induced obesity mouse model with supplementation for up to 16 weeks by measuring changes in body weight, glucose and insulin tolerance, and gut barrier function. We also utilized flow cytometry, quantitative PCR, enzyme-linked immunosorbent assay (ELISA), and histology to measure changes to immune cells populations and cytokine profiles in the intestine, visceral adipose tissue (VAT), and liver. 16SrRNA sequencing was performed to examine gut microbial differences induced by indigo supplementation. Results We identifed indigo, an aryl hydrocarbon receptor (AhR) ligand agonist, as a potent inducer of IL-10 and IL-22, which protects against high-fat diet (HFD)-induced insulin resistance and fatty liver disease in the diet-induced obesity model. Therapeutic actions were mechanistically linked to decreased inflammatory immune cell tone in the intestine, VAT and liver. Specifically, indigo increased Lactobacillus bacteria and elicited IL-22 production in the gut, which improved intestinal barrier permeability and reduced endotoxemia. These changes were associated with increased IL-10 production by immune cells residing in liver and VAT. Conclusions Indigo is a naturally occurring AhR ligand with anti-inflammatory properties that effectively protects against HFD-induced glucose dysregulation. Compounds derived from indigo or those with similar properties could represent novel therapies for diseases associated with obesity-related metabolic tissue inflammation. Introduction These authors contributed equally: Yi-Hsuan Lin, Helen Luck These co-senior authors contributed equally: Shawn Winer, Daniel A. Winer Supplementary information The online version of this article (https:// doi.org/10.1038/s41366-019-0340-1) contains supplementary material, which is available to authorized users. * Daniel A. Winer Extended author information available on the last page of the article Obesity is a major risk factor for type 2 diabetes and is associated with low-grade chronic inflammation [1]. The precursor to type 2 diabetes, known as insulin resistance (IR), can develop through multiple pathways, but the role of the immune system inside metabolic tissues and the intestine is an important contributor to this disease [2–6]. High-fat diet (HFD) changes the composition of the gut microbiota, leading to dysbiosis, which contributes to inflammatory immune changes in the bowel [7]. Leaked intestinal luminal components, such as lipopolysaccharides (LPS), reach the visceral adipose tissue (VAT) and other metabolic tissues including Y.-H. Lin et al. the liver, and promote local inflammation and insulin insensitivity [8–10]. During obesity, the expansion of VAT leads to adipocyte death resulting in the formation of immune cell crown-like structures (CLS) composed of accumulating immune cells [11, 12]. Pro-inflammatory cytokine secretion, including interferon (IFN)γ from CD8+ T cells and CD4+ Th1 T cells, and TNFα, interleukin (IL)-6 and IL-1β from recruited inflammatory (M1-like) polarized macrophages, can directly inhibit the insulin signaling pathway in tissues [11, 13, 14]. Regulatory T cells (Tregs) and resident antiinflammatory (M2-like) polarized macrophages that produce factors such as IL-10 help to maintain insulin sensitivity but are reduced within the VAT during obesity [15, 16]. The gut is home to the largest component of the body’s immune system known as the gut-associated lymphoid tissue. During homeostatic conditions, immune cells of the gut function to maintain the integrity of the intestinal barrier, promote tolerance to food antigens and commensal bacteria, and protect against invasive pathogens. Tregs and the production of IL-10 promotes mucin production by Goblet cells, and tolerance to the gut flora and dietary proteins [17, 18]. In addition, RORγt+ innate lymphoid cells (ILCs) and IL-22 producing CD4+ T cells can produce IL-22 to support the barrier function of intestinal epithelial cells (IECs) [19, 20]. One such mechanism is through the production of antimicrobial peptides (AMPs) by Paneth cells that regulate the gut microbiota and intestinal barrier integrity [21, 22]. In contrast, during obesity, hyperglycemia and proinflammatory mediators can disrupt intestinal barrier function, increase intestinal permeability, and lead to altered gut immunity with low levels of chronic inflammatory change [3, 23]. This compromised intestinal barrier results in bacterial product leakage and metabolic tissue inflammation, including increases in TNFα and reductions in IL-10 in VAT [2, 24]. In the gut, diet-induced obesity (DIO) in mice and humans leads to a Th1 response with increased IFNγ production coupled with a reduction in Th17 cells [4–6]. Furthermore, decreased intestinal ILC3s and IL-22producing CD4+ T cells also occur in HFD-fed mice which may result in less IL-22 production, worsening intestinal barrier function [3, 25]. Administration of exogenous IL-22 in HFD-fed mice improves the gut barrier, reduces LPS leakage and alleviates IR [25, 26]. Overall, IL22 production is critical for intestinal homeostasis and may be an important therapeutic target for obesity and IR. The aryl hydrocarbon receptor (AhR) is a transcription factor first identified for mediating the toxicity of environmental pollutants. Since then, AhR has also been discovered to be important for multiple physiological mechanisms, especially in the regulation of intestinal homeostasis. In metabolic syndrome, the production of AhR agonists by the gut microbiota is reduc (...truncated)